Simultaneous aspirator and dispenser for multiwell plates and similar devices

ABSTRACT

A method of making a measurement of binding affinity of a sample comprises the steps of: introducing the sample into a well having a bottom formed as a photonic crystal biosensor, wherein a portion of the sample becomes bound to the biosensor; making a measurement of the change in the shift in peak wavelength value as a function of time (k on ) from the well as the sample is introduced into the well; simultaneously dispensing the sample to the well and aspirating the sample from the well and measuring the change in the shift in peak wavelength value as a function of time (k off ) during the simultaneous dispensing and aspirating, and calculating an equilibrium association constant or an equilibrium dissociation constant for the sample from the values of k on  and k off .

CROSS-REFERENCE TO RELATED APPLICATION

This is a divisional of U.S. Ser. No. 11/601,020 filed Nov. 17, 2006,the content of which is incorporated by reference herein.

BACKGROUND

This disclosure relates to the art of devices used for aspirating anddispensing small quantities of fluids. More particularly, the disclosurerelates to a combination aspirating and dispensing device suitable foruse with test devices having a sample containment region in the form ofone or more wells or columns of wells, for example in the form of amulti-well plate.

Test devices in the field of biology and biochemistry can take a varietyof forms, including devices arranged in an array of wells, such as an8×12 array of wells arranged in rows and columns. In order to conduct atest on the sample the sample must be loaded into the well. A variety ofdispensing devices are known in the art, and described in the patentliterature. See, for example, U.S. Pat. Nos. 5,578,270; 6,374,683;6,325,114; 6,537,505 and 6,983,636. Some of these dispensing devices areautomated, while others require a human operator to manually dispense asample into a well of a test device.

The assignee of this invention has developed a grating-based biosensorwhich can be affixed to the bottom of a bottom-less multiwell platewhereby the multiwell plate forms a receptacle for holding a biochemicalsample to be tested. Grating-based sensors represent a new class ofoptical devices that have been enabled by recent advances insemiconductor fabrication tools with the ability to accurately depositand etch materials with precision less than 100 nm.

Several properties of photonic crystals make them ideal candidates forapplication as grating-type optical biosensors. First, thereflectance/transmittance behavior of a photonic crystal can be readilymanipulated by the adsorption of biological material such as proteins,DNA, cells, virus particles, and bacteria on the crystal. Other types ofbiological entities which can be detected include small and smallermolecular weight molecules (i.e., substances of molecular weight<1000Daltons (Da) and between 1000 Da to 10,000 Da), amino acids, nucleicacids, lipids, carbohydrates, nucleic acid polymers, viral particles,viral components and cellular components such as but not limited tovesicles, mitochondria, membranes, structural features, periplasm, orany extracts thereof. These types of materials have demonstrated theability to alter the optical path length of light passing through themby virtue of their finite dielectric permittivity. Second, thereflected/transmitted spectra of photonic crystals can be extremelynarrow, enabling high-resolution determination of shifts in theiroptical properties due to biochemical binding on the surface of thegrating while using simple illumination and detection apparatus. Third,photonic crystal structures can be designed to highly localizeelectromagnetic field propagation, so that a single photonic crystalsurface can be used to support, in parallel, the measurement of a largenumber of biochemical binding events without optical interferencebetween neighboring regions within <3-5 microns. Finally, a wide rangeof materials and fabrication methods can be employed to build practicalphotonic crystal devices with high surface/volume ratios, and thecapability for concentrating the electromagnetic field intensity inregions in contact with a biochemical test sample. The materials andfabrication methods can be selected to optimize high-volumemanufacturing using plastic-based materials or high-sensitivityperformance using semiconductor materials.

Representative examples of grating-type biosensors in the prior art aredisclosed in Cunningham, B. T., P. Li, B. Lin, and J. Pepper,Colorimetric resonant reflection as a direct biochemical assaytechnique. Sensors and Actuators B, 2002. 81: p. 316-328; Cunningham, B.T., J. Qiu, P. Li, J. Pepper, and B. Hugh, A plastic colorimetricresonant optical biosensor for multiparallel detection of label-freebiochemical interactions, Sensors and Actuators B, 2002. 85: p. 219-226;Haes, A. J. and R. P. V. Duyne, A Nanoscale Optical Biosensor:Sensitivity and Selectivity of an Approach Based on the LocalizedSurface Plasmon Resonance Spectroscopy of Triangular SilverNanoparticles. Journal of the American Chemical Society, 2002. 124: p.10596-10604.

The photonic crystal biosensors of the assignee and associated detectioninstruments for label-free binding detection are also described in thepatent literature; see U.S. patent application publications U.S.2003/0027327; 2002/0127565, 2003/0059855 and 2003/0032039. Methods fordetection of a shift in the resonant peak wavelength are taught in U.S.Patent application publication 2003/0077660. The above-references patentapplications and articles are hereby incorporated by reference in theirentirety.

There is currently a need in the art for simple, easy to use devicewhich allows for simultaneous dispensing and aspirating of a solutioncontaining a test sample onto a testing device, e.g., one configured inan array of wells. This invention meets that need.

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described andillustrated in conjunction with systems, tools and methods which aremeant to be exemplary and illustrative, not limiting in scope. Invarious embodiments one or more of the above-described problems havebeen reduced or eliminated, while other embodiments are directed toother improvements.

In a first aspect, a device is disclosed which provides forsimultaneously dispensing and aspirating a sample to a testing device.The testing device can take a variety of forms. The embodiments will bedescribed below in conjunction with a testing device in the form of amulti-well plate having a plurality of sample wells arranged in rows andcolumns. The principles of operation of the device are applicable toother types of testing devices.

The dispensing and aspirating device includes a body having a portionthereof, such as the bottom surface of the body, adapted for engagementwith the testing device. In the context of the multi-well test device,the bottom surface of the body rests on the top surface of themulti-well testing device when the dispensing and aspirating device isin use. The device further includes dispense tubing coupled to the bodyfor receiving a sample (e.g., fluid solution containing a sample) from asource. The device further includes aspirate tubing coupled to the bodywhich is connected to a source of vacuum. The device further includes adispense manifold connected to the dispense tubing having at least onedispensing port and an aspirate manifold connected to the aspiratetubing having at least one aspirating port.

The dispensing and aspirating device further includes a controlmechanism, e.g., in the form of one or more valves and button or switchfor operating the valves, for controlling movement of the sample fromthe dispense tubing to the dispense port and for simultaneouslycontrolling the application of vacuum in the aspirate tubing to theaspirating port. When the control mechanism is operated (e.g., bypressing on a button and responsively opening the valves), fluidsolution containing the sample is introduced into the dispense manifoldand exits the dispensing port whereby the sample is introduced into thetest device. Simultaneously, the vacuum is applied to the aspiratingport in the aspirate manifold and the sample which is applied to thetest device can be withdrawn.

As noted, the testing device may take the form of a multi-well testingdevice arranged in one or more rows of a plurality of wells. Thedispense and aspirate device is configured such that the dispense andaspirate manifolds include a dispensing and aspirating port for eachwell in the row of wells in the multi-well test device. Accordingly,when the control mechanism is activated, sample is introduced into eachwell in the row of wells and the sample solution is also aspirated fromeach of the wells in the row of wells.

The aspirating and dispensing device is particularly well suited fortesting devices that are configured in the form of a grating-basedbiosensor. The aspirating and dispensing device can be used to dispenseand aspirate a sample onto the biosensor surface while the detectioninstrument for the biosensor simultaneously operates to generate opticalmeasurements from the testing device, such as the shift in peakwavelength value due to binding of the sample to the surface of thebiosensor.

In another aspect, a method is disclosed for simultaneously dispensingand aspirating a sample to a testing device having at least one well.The method comprises the steps of: positioning an aspirating anddispensing device over the testing device such that an aspirating portand a dispensing port in the device are placed into the at least onewell, and activating a control mechanism to thereby cause a sample toenter the well via the dispensing port and simultaneously aspirating thesample from the well. In one embodiment, the testing device takes theform of a multi-well device having a plurality of wells arranged in oneor more rows of wells. During the positioning step an aspirating portand a dispensing port are placed into all the wells in one of the rowsof wells of the multi-well device.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by reference to thedrawings and by study of the following detailed descriptions.

BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary embodiments are illustrated in referenced figures of thedrawings. It is intended that the embodiments and figures disclosedherein are to be considered illustrative rather than restrictive.

FIG. 1 is a plan view of an embodiment of the dispensing and aspiratingdevice showing the device resting on the top surface of a testing devicein the form of a multi-well microtiter plate, with an aspirating portand a dispensing port positioned in each of the wells in a row of wellsin the multi-well plate for simultaneous dispensing and aspirating of asample into each sample well in a row of the wells.

FIG. 2 is a cross-sectional view of the device of FIG. 1, taken alongthe lines 2-2 of FIG. 1.

FIG. 3 is an end view of the device of FIGS. 1 and 2, shown isolatedfrom the testing device. The height of the dispense manifold and thedispense and aspiration ports are shown somewhat exaggerated in FIG. 3in order to show the structure of the device.

FIG. 4 is a graph of shift in peak wavelength value (PWV) (in nm) as afunction of time for a sample simultaneously dispensed into andaspirated from a well of the test device of FIGS. 1-3. FIG. 4 shows thePWV shifts for three different rates of sample dispensing andaspirating.

FIGS. 5-7 are plan, end and side views, respectively, of an alternativeembodiment of the aspirating and dispensing device of FIGS. 1-3, whichmay include one or more features including a gripping feature by whichthe device may be gripped by a robotic arm, sensors for sending chemicalproperties in the well such as pH or temperature, microfluidic valves,and an electrical connector for coupling the device to an electronicsunit for control by a robot, variable flow pump control, sensor signaloutput, and/or valve control.

FIG. 8 is a graph of shift in peak wavelength value (PWV) as a functionof time for a sample placed in a well of biosensor used with the deviceof FIG. 1-3 or 5-7, showing the acquisition of on- and off-rate dataindicating rates at which binding events occur in the biosensor wells,with the off-rate data acquired using simultaneous aspirating anddispensing features of the apparatus of FIGS. 1-3 or FIGS. 5-7.

DETAILED DESCRIPTION

FIG. 1 is a plan view of an embodiment of a dispensing and aspiratingdevice 10 showing the device 10 resting on the top surface 13 of atesting device 12 in the form of microtiter plate having wells 14arranged in a plurality of rows and columns. FIG. 2 shows the device 10in cross-section along the lines 2-2 of FIG. 1. FIG. 3 is an end view ofthe device 10 shown isolated from the testing device 12.

The dispensing and aspirating device 10 includes a body or enclosure 20which has bottom surface portion 15 which is given a substantiallyplanar configuration (best shown in FIGS. 2 and 3) so that the enclosure20 may rest upon and thereby engage the top surface 13 of the testingdevice 12 during use. The device 10 includes dispense supply tubing 22which is coupled to the body 20. The dispense supply tubing 22 carries afluid solution containing sample from a source (not shown) and deliversthe solution to the enclosure 20. The enclosure includes additionalconduits (shown in dashed lines in FIG. 2) and a dispense inlet tube 32for carrying the sample to a dispense manifold 40. A valve 30 (FIG. 2)or other flow regulation device is placed within the enclosure 20 toregulate the flow of the sample solution to the dispense manifold 40.

Dispense return tubing 24 is also coupled to the body 20. The dispensereturn tubing 24 allows for a continuous loop of the dispensing solutionthrough the device and back to the solution reservoir connected to thesupply tubing 22 at a constant flow rate and pressure until the valve 30is opened and the solution is able to travel into the wells 14. Thisloop consisting of supply tubing 22 and return tubing 24 allows for apump to be attached to the device and connected to the tubing and thusproviding the constant flow and pressure. If there was no loop, the flowrate would accelerate from 0 to a set value as the solution is allowedto enter the wells. The pressure would also decrease after being allowedby the valve to enter the wells.

The body 20 is also coupled to aspirate tubing 26 which is connected toa source of vacuum. The aspirate tubing 26 is connected to internalconduits which supply the vacuum to an aspirate manifold 42. A valve orlike device is placed within the body 20 to regulate the application ofvacuum to the aspirate manifold 42.

The dispense manifold 40 includes at least one dispensing port 46. Inthe embodiment of FIGS. 1-3, there are eight dispensing ports 46connected to the dispense manifold, one for each well in a row of wellsin the testing device 12.

Similarly, the aspirate manifold 42 includes at least one aspiratingport 48. In the embodiment of FIGS. 1-3, there are eight aspiratingports 48 connected to the aspirate manifold 42, one for each well in arow of wells. The aspirate outlet 34 couples the aspirate manifold 42 tothe valve 30 connected to the aspirate tubing 26.

As will be appreciated from FIGS. 2 and 3, the ports 46 and 48 arepositioned in close proximity to each other and grouped in pairs, suchthat one dispensing port and one aspirating port are positioned withineach well across the row of wells 14.

The aspirate and dispense manifolds 40 and 42 include removable end caps44 which allow for cleaning and disinfection of the interior of themanifolds 40 and 42 after use.

The device 10 further includes a control mechanism for controllingmovement of the sample from the dispense tubing 22 to the dispense ports46 and for simultaneously controlling application of vacuum in theaspirate tubing 26 to the aspirating ports 48. In one embodiment shownin FIGS. 1-3, the control mechanism takes the form of a manuallyactuated button 28 positioned on the top surface of the enclosure orbody 20. When the button 28 is depressed, a valve 30 (FIG. 2) in theenclosure 20 is opened to cause solution (under positive pressure) inthe dispense supply tubing 22 to enter and fill the dispense manifold 40and exit the dispense ports 46 into each of the wells of a row of wellsin the microtiter plate 12. Simultaneously, a second valve 30 is openedallowing vacuum present in the aspirate tubing 26 to be applied to theaspirate manifold 42. The vacuum is present at the tip of the aspirateports 48, which then withdraws the solution from the wells.Consequently, by virtue of the design shown in FIGS. 1-3, with anaspirating port and a dispensing port positioned in each of the wells ina row of wells in the multi-well testing device 12, when the controlmechanism or button 28 is depressed, the device 10 simultaneouslydispenses a sample into each sample well in a row of the wells andaspirates the sample from the each well.

While the are eight aspirating ports and eight dispensing ports in theembodiment of FIGS. 1-3, arranged in pairs as shown in FIG. 3, thenumber of ports can of course be increased or decreased as necessary toaccommodate testing devices with different numbers of wells in a row ofwells.

In order to dispense and aspirate solution containing a sample to allthe wells in the row simultaneously, the aspirate and dispense manifolds40 and 42 are given an elongate tubular channel configuration shown inFIG. 3 having a length L. Additionally, the testing device 12 consistsof an array of wells 14 arranged in rows and columns, and wherein atleast one of the rows and columns of wells is of a linear dimension M(see FIG. 1), and where L≧M. Additionally, there is one aspirating port48 and one dispensing port 46 in a spaced relation along the length ofthe aspirate and dispense manifolds 42 and 40 for each well 14 in therows of wells of length M, as shown in FIGS. 1-3.

In the embodiment of FIGS. 1-3, the body 20 is sized and shaped so as tobe held in a human hand, e.g., the body can be shaped similar to acomputer mouse. The manually-activated control device 28 is incorporatedinto the body 20, e.g., on the top of the body 20 where it can bedepressed by the index finger. The user operates the control device 28to dispense and aspirate solution into all the wells in one rowsimultaneously, then lifts the device 10 from the testing device 12 andpositions the ports 46 and 48 into the wells of the next row of wells,activates the control mechanism or button 28, and then repeats theprocess for the remaining rows of wells. It will be noted from FIG. 2that the tips of the dispensing and aspirating ports 46 and 48 extendinto wells 14 formed in the top surface 13 of the testing device 12 whenthe lower surface 15 of the body 20 is engaged with the top surface 13of the testing device.

In the embodiment of FIGS. 1-3, the device 10 further includes anauxiliary injection port 36 for receiving a second sample forintroduction to the testing device 12.

The device 10 of FIGS. 1-3 is particularly well suited for use inconjunction with testing devices 12 which include plurality of wells forreceiving the sample, and in which the wells have a bottom surface forreceiving the sample constructed as a grating-based biosensor 50 (FIG.2). See the references disclosed in the Background section as examplesof such grating-based biosensors. One virtue of the present dispensingand aspirating device 10 is that that the device may be operated todispense and aspirate solution containing a biochemical sample to thetesting device while the testing device is being read by opticaldetection instrumentation. Accordingly, the testing device 12 can beused to detect binding interactions on the surface of the biosensor 50(e.g., shifts in PWV), at different rates for dispensing and aspiration.For example, FIG. 4 is a graph of shift in peak wavelength value (PWV)(in nm) as a function of time for a sample introduced into a well of thetest device of FIGS. 1-3 that is configured as a grating-basedbiosensor. FIG. 4 shows the PWV shifts for three different rates ofsample dispensing and simultaneous aspiration.

As noted above, the control mechanism 28 in one embodiment is manuallyoperated, by the user of the device manually depressing the dispensingbutton 28 to thereby open the valves and allow the dispensing andaspirating to occur. The dispensing and aspirating continues as long asthe button 28 is held down.

In one possible variation, the entire dispensing and aspirating device10 could be designed for an automated system automatically dispensingand aspirating solution containing a sample into a testing device, inwhich situation the control mechanism 28 could by automaticallyoperated. FIGS. 5-7 show an alternative configuration designed forautomatic operation, e.g., with movement by a robotic and control viaelectronic controls. In this embodiment, the body 20 includes a pair ofrobot gripping features, e.g., slots 62 formed in the side of the bodyby which a robotic hand or arm may grip the device, lift it up and placeit down, so that the probes are sequentially placed in rows of wells inthe test device. The particular type of gripping feature in the body 20is not particularly critical and can be adapted to the particular handor arm construction of the robot being used with the device. The body 20further includes an electrical connector 60 with a set of pins forconnection to wires leading to electronic controls for the valve(s) 30(FIG. 2) in the body. In this case, the valve(s) are electricallyoperated and open and close in response to signals supplied to the bodyvia the electrical connector 30. The electrical connector will alsoinclude pins for variable flow pump control, and output of signals frompH, temperature, or other sensors 66.

In this embodiment, the device 10 could be attached to a robotic arm(not shown) which lifts the device 10 into and out of engagement withthe top surface of the testing device 12. The operation of thedispensing and aspirating mechanisms could be performed by switching onand off electrically-operated valves which are either built into thedevice 10 or which are otherwise in the fluid path between the source ofsolution and the dispensing ports and between the source of vacuum andthe aspirating ports. Persons skilled in the art can readily adapt thedisclosed embodiment to an automated dispensing and aspiratingembodiment without undue difficulty, given the state of the art ofrobotics and electronic control systems.

In another variation of the embodiment of FIGS. 1-3, an electroniccontrol mechanism from the aspirating and dispensing device may operatea variable flow pump that directs sample to the testing device 12,either in addition to a valve 30 or in lieu of the valve 30. Thevariable flow pump could be positioned upstream of the device 10, butoperated by actuation of a button or other manual control incorporatedinto the device 10. For example, the device 10 could include a dial,thumb wheel, or other type of manual control which adjusts the settingof the variable flow pump. Such control feature may also include a dialto display to the operator the current setting of the variable flowpump.

In another possible variation, the device 10 can further include one ormore sensors 66 (FIG. 7) for making a measurement of a sample deliveredby the aspirating and dispensing device to the wells 14 of the testingdevice. For example, a sensor 66 such as a temperature sensor, pHsensor, or an ionic strength sensor could be incorporated into thedevice and positioned or mounted adjacent to at least one of theaspirating port 46 or the dispensing port 48, as shown in FIG. 7. Morethan one type of sensor could be incorporated. Furthermore, thesensor(s) could be provided for each pair of dispensing and aspiratingports, or just for one pair of dispensing and aspirating ports. Theoutput of the sensors is supplied to the control electronics (not shown)via the connector 60 of FIG. 5. For example, the sensors 66 can becoupled to a processing unit (not shown) that controls delivery of thesample to the testing device 12 using a feedback loop incorporatingsensor data reported by the at least one sensor. For example, if thedevice includes a pH sensor, the processing unit adjusts the pressure inthe dispense supply tubing 22 according to the pH reading provided bythe pH sensor.

In still another variation, the aspirating and dispensing device 10 mayfurther be associated with a temperature controller (not shown) whichcontrols the temperature of the sample delivered to the testing device12. The temperature controller may include a temperature sensor (eitherthe sensor 66 in the well 14 or in the body 20 or elsewhere) and aheating element or cooling element to heat or cool the sample as neededso that the sample is introduced to the wells of the testing device 12at a desire temperature or in accordance with a desired temperatureprofile.

As shown in FIGS. 5-7, the aspirating and dispensing device furtherincludes eight microfluidic valves 64 which are placed in the dispensemanifold 40 and control dispensing of sample into the eight wells in arow of wells 14 in the test device 12. The microfluidic valves 64 can beeither mechanically or electrically operated, e.g., in response todepression of the button 28 or by signals supplied to the device via theconnector 60.

In view of the above, it will also be appreciated that we have discloseda method of simultaneously dispensing and aspirating a sample to atesting device 12 having at least one well 14, comprising the steps of:positioning an aspirating and dispensing device 10 over the testingdevice 12 such that an aspirating port 48 and a dispensing port 46 inthe device 10 are placed into the at least one well 14; and activating acontrol mechanism (28) to thereby cause a sample to enter the well 14via the dispensing port 46 and simultaneously aspirating the sample fromthe well via the aspirating port 48. In one configuration, as shown inFIGS. 1 and 2, the testing device 12 comprises a multi-well device(e.g., 96 well microtiter plate) having a plurality of wells 14 arrangedin rows and columns, and wherein during the positioning step anaspirating port 48 and a dispensing port 46 are placed into all thewells in a row or column of wells of the multi-well device 12.

As noted above, the activating step can be performed manually. It canalso be performed automatically, e.g., in an automated implementation ofthe device 10.

As noted further, the method may further involve making measurements ofthe sample with a sensor. The sensor may for example take the form of atemperature sensor, a pH sensor, or an ionic strength sensor. All threesensors may be incorporated into the device.

In one configuration, the sensor is a temperature sensor and the methodfurther includes the step of controlling the temperature of the sampledelivered to the testing device 12.

In one possible use of the device 10, the aspirating and dispensingsteps may be performed simultaneously with optical measurements taken ofthe testing device, e.g., measurements of shift in PWV indicatingbinding events occurring on the surface of the testing device. Forexample, FIG. 4 shows a graph of shift of PWV of a Mouse IgG sample as afunction of time at three different sample dispensing rates. The data iscaptured by an imaging instrument as disclosed in the above-referencesSRU Biosystems, Inc. published US patent application documents.

Measurements of Equilibrium Dissociation and Association Constants UsingDevice 10

The device 10 of FIGS. 1-3 and 5-7 can be used to measuring equilibriumdissociation and association constants of a sample being tested. Onetraditional biochemical definition of affinity between two moleculesinvolves the measurement of the equilibrium dissociation constant or theequilibrium association constant. These can be defined by the followingequations:

K _(d) =k _(off) /k _(on)   1)

and

K _(a) =k _(on) /k _(off)   2)

where

K_(d) is the equilibrium dissociation constant;K_(a) is the equilibrium association constant;k_(off) is the rate that the two molecules come apart;k_(on) is the rate that the two molecules bind together.

The device 10 of this disclosure is suitable for measurement of theconstants K_(a) and K_(d). In particular, the device 10 allows one toacquire off-rate data on a multi-well plate biosensor without having tomanually aspirate and dispense a solution with a handheld multi-channelpipettor. The device 10 allows for a constant simultaneous flow ofsolution into and out of the wells while data acquisition is occurring.This is not feasible with a hand-held pipettor since the flow rates arenot capable of precise control with hand-operated instruments such as apipettor. However, precise flow rates are possible with the aspiratingand dispensing device 10 of this disclosure.

The steps to make the on- and off-rate measurements and determination ofconstants K_(a) and K_(d) will now be explained in conjunction withFIGS. 1 and 8. A sample holder is used in the form of a multi-wellplate. 12 with the bottom of the wells 14 of the plate 12 formed as aphotonic crystal biosensor 50 as described above and in the patentapplications of the assignee SRU Biosystems cited previously in thisdocument. A buffer solution is added to one of the wells of thebiosensor. The biosensor is placed onto an detection instrument fordetecting the peak wavelength value of light reflected from the surfaceof the biosensor as described in the above-cited SRU published patentapplication documents. A baseline PWV of buffer is measured by thedetection instrument. See FIG. 8, curve between time 10 minutes and timeT1. Next, a ligand is then added to the well of the biosensor and mixedwith the buffer. This occurs at time T1 in FIG. 8.

The detection instrument continues to make measurements of the shift inthe PWV as the sample is added. There is an increase in the PWV signal,as indicated in FIG. 8 at 72. The on-rate (k_(on)) is the measurement ofthe change in Shift vs the change in Time, i.e., the slope of the curveof FIG. 8 at region 72.

After a period of time the ligand comes to equilibrium and the PWV shiftplateaus. See FIG. 8, point 73 and region 74.

At time T2, the Simultaneous Aspirator and Dispenser 10 (FIG. 1) is thenplaced on the sensor with a pair of the aspiration and dispenser probesplaced into the well containing the buffer+ligand. The valve 30 in thedispenser 10 is opened in order to start the flow of buffer into thewells via the dispense probe 46 (FIG. 2). Aspiration of buffer via theaspiration probe 48 (FIG. 2) is also activated in order to keep thebuffer at a steady flow over the sensor, with the rate of aspirationequal to the rate of dispensing.

As indicated at FIG. 8 at 76 and 78, the detection instrument records adecrease in PWV signal, as there is now a new equilibrium state for theligand with a change in the buffer in the well. This negative change inPWV Shift as a function of time is the off-rate (k_(off)), i.e., theslope of the curve at point 78. Eventually the shift in PWV plateaus asindicated at 80.

From the measurements of k_(on) and k_(off) one can compute theequilibrium disassociation and association constants using equations 1)and 2).

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions and sub-combinations as are within their truespirit and scope.

1. A method of making a measurement of binding affinity of a sample,comprising the steps of: introducing the sample into a well having abottom formed as a photonic crystal biosensor, wherein a portion of thesample becomes bound to the biosensor; making a measurement of thechange in the shift in peak wavelength value as a function of time(k_(on)) from the well as the sample is introduced into the well;simultaneously dispensing the sample to the well and aspirating thesample from the well and measuring the change in the shift in peakwavelength value as a function of time (k_(off)) during the simultaneousdispensing and aspirating, calculating an equilibrium associationconstant or an equilibrium dissociation constant for the sample from thevalues of k_(on) and k_(off).
 2. The method of claim 1, wherein the stepof simultaneously aspirating and dispensing is performed by a devicecomprising: a) a body having a portion thereof adapted for engagementwith a testing device incorporating the well; b) dispense tubing coupledto the body for receiving a sample from a source; c) aspirate tubingcoupled to the body connected to a source of vacuum; d) a dispensemanifold connected to the dispense tubing having at least one dispensingport; e) an aspirate manifold connected to the aspirate tubing having atleast one aspirating port; and f) a control mechanism for controllingmovement of the sample from the dispense tubing to the dispense port andfor simultaneously controlling application of vacuum in the aspiratetubing to the aspirating port.
 3. The method of claim 2, wherein testingdevice comprises a multi-well testing device arranged in one or morerows of a plurality of wells, and wherein the dispense and aspiratemanifolds include a dispensing and aspirating port for each well in therow of wells in the multi-well test device.
 4. The method of claim 1,further comprising the step of making a measurement with a sensor duringthe aspirating and dispensing step.
 5. The method of claim 4, whereinthe sensor is selected from the group of sensors consisting of a) atemperature sensor, b) a pH sensor, and c) an ionic strength sensor. 6.The method of claim 1, further comprising the step of controlling thetemperature of the sample delivered to the testing device.
 7. The methodof claim 1, wherein the well is incorporated into a testing device, andwherein the step of simultaneously dispensing and aspirating the samplecomprises the steps of: positioning an aspirating and dispensing devicehaving an aspirating port and a dispensing port proximate to the testingdevice such that the aspirating port and the dispensing port in thedevice are placed into the well; and activating a control mechanism tothereby cause a sample to enter the well via the dispensing port andsimultaneously aspirating the sample from the well.
 8. The method ofclaim 7, wherein the testing device comprises a multi-well device havinga plurality of wells arranged in rows and columns, and wherein duringthe positioning step an aspirating port and a dispensing port of theaspirating and dispensing device are placed into all the wells in a rowor column of wells of the multi-well device.
 9. The method of claim 1,further comprising the step of making an optical measurement of duringthe dispensing and aspirating step.
 10. The method of claim 1, whereinthe sample comprises a buffer.
 11. The method of claim 10, furthercomprises the step of making a measurement of a shift in peak wavelengthvalue of the biosensor during the step of dispensing and aspirating thebuffer.
 12. The method of claim 1, wherein the sample comprises a fluidsample containing at least one of the following: proteins, DNA, cells,virus particles, bacteria, low molecular weight molecules (substances ofmolecular weight<1000 Daltons (Da)), molecules having a molecular weightof between 1000 Da to 10,000 Da, amino acids, nucleic acids, lipids,carbohydrates, nucleic acid polymers, viral particles, viral components,and cellular components comprising vesicles, mitochondria, membranes,structural features, periplasm or an extract thereof.